Figure 1.

Generation of recombinant vesicular stomatitis virus (VSV) expressing the C-terminal 19 amino acid deletion SARS-CoV-2 spike mutant (VSVΔG-SΔ19) with replication capability. (A) Schematic representation of the genomic organization of the G protein-deficient VSV vector (VSVΔG) inserted with the C-terminal 19 amino acid truncated SARS-CoV-2 spike protein (SΔ19). N, nucleoprotein; P, phosphoprotein; M, matrix; GFP, green fluorescent protein; L, large polymerase. (B, C) Vero E6, HEK293T-hACE2, and BHK21-hACE2 cells were infected with the VSVΔG-SΔ19 virus (early passage, moi=0.5). Virus-driven GFP expression was monitored by fluorescence microscopy at 24 hrs post-infection (B). The SΔ19 protein expression in cells (left panel) and supernatant (right panel) was examined by immunoblotting (C). (D) The VSVΔG-SΔ19 virus was propagated in HEK293T-hACE2 cells, and the virus titer was determined in BHK21-hACE2 cells. (E) After a few passages in HEK293T-hACE2 cells, the viruses were inoculated in BHK21-hACE2 cells with limiting dilution. The replication-competent VSVΔG-SΔ19 viruses (SΔ19 Rep) emerging from BHK21-hACE2 cells were observed with GFP monitoring. (F) The S protein expression on recombinant virus particles of VSVΔG/G, the early passage of VSVΔG-SΔ19 enveloped with the VSV glycoprotein (SΔ19/G; replication incompetent) and SΔ19 Rep virus was examined by immunoblotting. (G, H) Vero E6 cells were infected with the SΔ19 Rep virus (moi=1, n=3). Virus titer (G) and S protein expression on virus particles (H) in culture supernatant were examined. moi, multiplicity of infection; dpi, days post-infection. Scale bar: 100 µm.